G protein-coupled receptor antagonist and its use for preventing and treating alzheimer&#39;s disease

ABSTRACT

The invention discloses methods for screening a reagent for treating or preventing Alzheimer&#39;s disease or related neurological pathology. A method according to the invention includes the steps of: (a) activating a receptor and determining a first extent of endocytosis of the receptor, wherein the receptor is a G-protein coupled receptor that associates with presenilin-1; (b) activating the receptor under the same conditions as in step (a), in the presence of a candidate reagent, and determining a second extent of endocytosis of the receptor; (c) determining a difference between the first extent of endocytosis and the second extent of endocytosis; and (d) repeating steps (a)-(c), if the difference is less than a threshold. The invention also disclose uses of receptor antagonists for manufacturing medicaments for treating or preventing Alzheimer&#39;s disease or related neurological pathology, wherein the receptor antagonists inhibit endocytosis of a G-protein coupled receptor that associates with presenilin-1 during endocytosis.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a divisional application of application Ser. No. 12/159,183,filed on Jun. 25, 2008, which is a national stage application ofPCT/CN2006/003595, filed on Dec. 26, 2006, which claims priorities toChinese application No. 200610162480.7, filed on Nov. 17, 2006, andChinese application No. 200510112005.4, filed on Dec. 26, 20057, 2006.The present application claims priorities to all these priorapplications and incorporates these prior applications by reference intheir entirety.

BACKGROUND OF INVENTION

1. Field of the Invention

The present invention relates generally to prevention or treatment ofAlzheimer's disease or related neurological pathologies; particularly,it relates to the use of β-adrenergic or opioid receptor antagonists fortreating Alzheimer's diseases.

2. Background Art

Alzheimer's Disease (AD), which is characterized by progressive dementiaand personality dysfunction, is the most common neurodegenerativedisorder associated with aging. AD affects 5-11% of the population overthe age of 65 and 30% of those over the age of 85. AD is caused byabnormal accumulation of amyloid plaques in the vicinity of degeneratingneurons and reactive astrocytes. D. J. Selkoe, Annu. Rev. Neurosci. 17,489 (1994).

Amyloid plaque, composed mostly of amyloid β (Aβ), is a hallmark of ADneuropathology, and formation of amyloid plaques is considered a primarycause of AD. In addition, recent studies have revealed that thediffusible oligomeric AP may also be neurotoxic and potentiallyAD-related. Walsh, D. M. et al., “Naturally Secreted Oligomers ofAmyloid Beta Protein Potently Inhibit Hippocampal Long-Term Potentiationin vivo,” Nature 416, 535-9 (2002)

Aβ is generated from β-amyloid precursor protein (APP) via sequentialcleavages by β- and γ-secretases. As illustrated in FIG. 1, APP iscleaved by β-secretase to produce a soluble APP_(S)-β fragment and a C99fragment, the latter is in turn cleaved by γ-secretase to produce the Aβfragment and a C60 fragment.

The amyloid fragments (Aβ) comprise at least two forms, a 40 amino acidform (Aβ₄₀) and a 42 amino acid form (Aβ₄₂). The 42 amino acid form(Aβ₄₂) is more prone to plague formation and is considered more relevantto AD etiology. γ-Secretase plays a pivotal role in AD because itdetermines the ratio of the two main Aβ species (Aβ₄₀ and Aβ₄₂).

As shown in FIG. 2, γ-secretase complex includes at least four essentialcomponents: presenilin (PS), nicastrin (NCSTN), APH-1, and PEN-2.Mutations in the putative catalytic component presenilin-1 (PS1) accountfor most cases of familial AD (FAD), suggesting that γ-secretase may becritically involved in the pathogenesis of AD (at least the pathogenesisof FAD).

Although the correlation between presenilin-1 mutations and FAD providesa clue to genetic cause of AD, FAD accounts for less than 10% of all ADcases. In contrast, most AD cases are sporadic in nature, indicatingthat factors other than presenilin-1 mutations are more important in thepathogenesis of AD. Therefore, it is important to investigate how otherfactors or environmental influences contribute to AD pathogenesis.

Previous studies have shown that Aβ production in cell cultures can bereduced by activation of intracellular signaling pathways or membranereceptors such as muscarinic acetylcholine receptors. Recent evidencealso shows that Aβ levels and amyloid plaque formation can be influencedby somatostatin or environmental factors.

APP processing is also regulated by neurotransmitters and synapticactivity. For example, activation of neurotransmitter receptors, whichare coupled to phosphotidylinositol (PI) hydrolysis or to protein kinaseC (PKC) activation, can promote APP metabolism and decrease amyloidformation. (Ulus and Wurtman, J. Pharm. Exp. Ther., 281,149 (1997)) Onthe other hand, activation of neurotransmitters coupled to cAMPproduction can suppresses both constitutive and PKC/PI-stimulated APPssecretion in astroglioma cells and in primary astrocytes. (Lee et al.,J. Neurochem., 68,1830 (1997)) The inhibitory effect of cAMP on APPssecretion may be specific for astrocytic cells because cAMP and PKAactivation reportedly stimulate APPs secretion in pheochromocytoma PC-12and human embryonic kidney cells. (Xu et al., PNAS USA, 93, 4081 (1996);Marambaud et al., J. Neurochem., 67, 2616 (1996)) In any event, theabove results indicate that changes in neurotransmitter levels or secondmessenger signaling, which may result from neuron degeneration andsynapse loss in AD, can disrupt APP processing and lead to accumulationof amyloidogenic or neurotoxic APP fragments.

Furthermore, it has been shown that modulation of β-adrenergicreceptors, which leads to elevated cAMP, can increase the synthesis ofAPP in astrocites. Based on this finding, U.S. Pat. Nos. 6,187,756 and6,043,224, issued to Lee et al., discloses methods for alleviatingneurological disorders stemming from the aberrant expression of APP byusing β-adrenergic receptor antagonists that modulate the cAMP levels.In this approach, β-adrenergic receptor antagonists are used to suppressthe synthesis of APP, through modulation of cAMP levels.

In addition to suppression of APP synthesis, modulation of APPmetabolism may also be used to alleviate neurological disordersassociated with APP-related plaque formation. For example, U.S. Pat. No.5,385,915,issued to Buxbaum et al., discloses methods and compositionsfor affecting APP processing using agents that regulate proteinphosphorylation, i.e., agents that affect kinases or phosphatases. Themodulation of APP processing in turn leads to the regulation of theproduction of Aβ peptides that accumulates in amyloidogenic plaques.

Similarly, in U.S. Pat. No. 5,242,932, Gandy et al. disclose and claim amethod of modulating or affecting the intracellular trafficking andprocessing of proteins (including APP) in mammalian cells, usingchemicals such as chloroquine and primaquine.

While these prior art methods seem to be effective in modulating theproduction and metabolism of APP, and hence the formation of plaques,there remains a need for more methods and reagents for the treatment andprevention of AD.

SUMMARY OF INVENTION

Objectives of the present invention include providing methods forscreening reagents for treating or preventing Alzheimer's disease orrelated neurological pathology, thereby providing reagents for treatingor preventing Alzheimer's disease or related neurological pathology.

In one aspect, embodiments of the invention relate to methods forscreening a reagent for treating or preventing Alzheimer's disease orrelated neurological pathology. A method in accordance with oneembodiment of the invention includes the steps of: (a) activating areceptor and determining a first extent of endocytosis of the receptor,wherein the receptor is a G-protein coupled receptor that associateswith presenilin-1; (b) activating the receptor under the same conditionsas in step (a), in the presence of a candidate reagent, and determininga second extent of endocytosis of the receptor; (c) determining adifference between the first extent of endocytosis and the second extentof endocytosis; and (d) repeating steps (a)-(c), if the difference isless than a threshold.

Another aspect of the invention relates to methods for screening areagent for treating or preventing Alzheimer's disease or relatedneurological pathology. A method in accordance with one embodiment ofthe invention includes the steps of: (a) measuring a first extent ofassociation between a receptor and presinilin-1 or γ-secretase, whereinthe receptor is a G-protein coupled receptor that associates withpresenilin-1; (b) measuring a second extent of association between thereceptor and presenilin-1 or γ-secretase, under the same conditions asin step (a), in the presence of a candidate reagent; (c) determining adifference between the first extent of association and the second extentof association; and (d) repeating steps (a)-(c), if the difference isless than a threshold.

Another aspect of the invention relates to uses of receptor antagonistsfor manufacturing medicaments for treating or preventing Alzheimer'sdisease or related neurological pathology. A use in accordance with oneembodiment of the invention is characterized in that the receptorantagonist inhibits endocytosis of a G-protein coupled receptor thatassociates with presenilin-1 during endocytosis.

Other aspects and advantages of the invention will become apparent fromthe following description and attached claims.

BRIEF SUMMARY OF DRAWINGS

FIG. 1 illustrates the process of sequential actions of β-secretase andγ-secretase in the formation of Aβ from APP. APP is first cleaved byβ-secretase to generate a soluble APPs-β and C99. C99 is in turn cleavedby γ-secretase to generate Aβ and C60.

FIG. 2 shows the four main components of γ-secretase: presenilin,nicastrin (NCSTN), ACH-1, and PEN-2.

FIG. 3 illustrates a process of endocytosis as a result of receptoractivation and trafficking of the endocytosed vesicles to LEL.

FIG. 4 shows a flow chart of one method in accordance with oneembodiment of the invention for screening a receptor antagonist for thetreatment or prevention of Alzheimer's disease.

FIGS. 5 a-5 e show GPCR stimulation increases Aβ production in celllines and primary hippocampal cells. HEK293 cells co-expressing β₂AR andAPPswe (a) were treated for 1 h with 10 μM Iso in the absence orpresence of 10 μM 4 Pro after 6-h pre-treatment of vehicle or 1 μML685,458; HEK293 cells co-expressing β₂AR and C99 (b to c) were treatedfor 1 h with 10 μM Iso in the absence or presence of 10 μM Pro. Primaryhippocampal cultures expressing C99 (d) were treated for 1 h with 10 μMIso or 1 μM DADLE. Secreted Aβ₄₀ (dark bars) and Aβ₄₂ (light bars) weredetected by ELISA, and their values are the mean±S.E. of threeindependent experiments and presented as fold values of the basal levels(*P<0.01). (e) Pulse-chase analysis of C99 cleavage in DOR and C99co-transfected HEK293 cells with or without 1-h treatment of 1 μM DADLE.The data are representative of at least three independent experiments.Con, control; Iso, isoproterenol; Pro, propranolol; DADLE, [D-Ala2,D-Leu5]-Enkephalin. Pro, propranolol; DADLE, [D-Ala²,D-Leu⁵]-enkephalin; NALT, naltrindole.

FIGS. 6 a-6 d show β₂AR stimulation enhances γ-secretase activity inneuronal cells. (a) HEK293 cells co-transfected with C99 and β₂AR weretreated for different time with 10 μM Iso in the absence or presence of10 μM Pro. The cell membrane fractions were incubated in vitro at 37° C.for 2 h and the generated C60 from these cell membrane fractions weredetected by Western blot. The data are representative of at least fourindependent experiments. (b to d) C6 glioma (b), rat acute hippocampalslices (c), and β₂AR-transfected wild-type and presenilin-deficient MEFs(d) were treated for 30 min with the indicated reagents including 10 μMIso, 10 μM Pro, 1 μM DADLE and 1 μM NALT respectively. The membranefractions from cells or slice homogenates were subjected to fluorogenicsubstrate assay. Data are means±S.E. of at least three independentexperiments and presented as fold values of the basal activity(*P<0.001). NALT, naltrindole.

FIGS. 7A-7B show results of enhanced γ-secretase activity as a result ofDOR activation, and time course of such enhancement. SH-SY5Yneuroblastoma (FIG. 7A) and acute hippocampal slices (FIG. 7B) weretreated with 1 μM DADLE or 1 μM NALT for 30 minutes. The membranefractions were subjected to fluorogenic substrate assay (*P<0.001). FIG.7C shows time course of g-secretase activity after activation of β2AR.C6 glioma were stimulated with 10 μM Iso for the indicated time. Thecell membrane fractions were subjected to fluorogenic substrate assay(*P<0.01).

FIGS. 8 a-8 g show receptor endocytosis associates with enhancedγ-secretase activity. (a) HEK293 cells were transfected with β₂AR orβ₂AR TYY and treated with 10 μM Iso for 30 min; (b) C6 glioma weretreated for 30 min with the indicated reagents including 10 μM Iso, 1μg/ml CTX, 10 μM Fsk, and 1 mM db-cAMP (left); (c) C6 glioma weretreated for 30 min with 10 μM Iso after pretreatment with the indicatedreagents including: 0.25 mg/ml Con A, 0.5 M Suc, and potassium depletedmedium. (d) C6 glioma were transfected with β-gal or Dyn K44A andtreated with 10 μM Iso for 30 min; (e) HEK293 cells were transfectedwith NS or clathrin RNAi and treated with 10 μM Iso for 30 min. The cellmembrane fractions in (a to e) were subjected to fluorogenic substrateassay. (f and g) HEK293 cells transfected with the indicated receptorswere treated for 30 min with 10 μM Iso and subjected toimmunofluorescence assay (f) or fluorogenic substrate assay (g). Data in(a to e and g) are means±S.E. of at least three independent experimentsand presented as fold values of the basal activity (*P<0.001). CTX,cholera toxin; Fsk, forskolin; db-cAMP, dybutyl cyclic adenosinemonophosphate; PTX, pertussis toxin; Dyn K44A, dynamin II K44A; Con A,concanavalin A; Suc, hypertonic sucrose solution; K⁺ dpl,potassium-depleted medium; NS RNAi, nonspecific RNA interference; β₂ARm,β₂AR L339,340A.

FIG. 9 shows the enhanced γ-secretase activity by DOR activation is notabolished by PTX treatment. SH-SY5Y neuroblastoma were stimulated with 1μM DADLE after 12 hours pre-treatment with 200 ng/ml PTX. The cellmembrane fractions were subjected to fluorogenic substrate assay(*P<0.01).

FIG. 10 shows receptor endocytosis induced by transferrin does not leadto enhanced γ-secretase activity.

FIGS. 11 a-11 d show increased γ-secretase and Aβ in endocyticcompartments. (a) HEK293 cells co-transfected with β₂AR and theindicated plasmids in combination with or without C99, were treated with10 μM Iso. The membrane and cytoplasma fractions were subjected tofluorogenic substrate assay (*P<0.001) or Western blot, respectively.(b) HEK293 cells were transfected and treated as in (a). The cytoplasmafractions were subjected to Aβ-specific immunoprecipitation/Westernblot. (c) HEK293 cells co-transfected with β₂AR, C99 and Flag-Rab7, weretreated for 1 h with 10 μM Iso. The cell homogenates were subjected toimmuno-isolation of LEL and Western blot using specific antibodies forthe indicated proteins. Data are representative of at least threeindependent experiments. (d) HEK293 cells co-transfected with HA-β₂ARand GFP-Rab7 were treated for 30 min with 10 μM Iso and detected for thedistribution of PS1 (red), GFP-Rab7 (green), and β₂AR (blue). Arrowsmark the punctual structures containing PS1 and GFP-Rab7.

FIG. 12 shows the enhanced γ-secretase activity by DOR activation isalso associated with LEL fractions.

FIGS. 13 a-13 d show γ-secretase enrichment requires endocytictransport. (a) HEK293 cells transfected with HA-β₂AR and Flag-Rab7 incombination with β-gal, Dyn K44A or Rab5 S34N, were treated with 10 μMIso for 30 min, stained with antibodies against PS1-NTF (red) or Flag(green). Scale bar, 8 μm. (b) HEK293 cells transfected with HA-DOR weretreated with 1 μM DADLE for 3 min, stained with antibodies againstPS1-NTF (red), DOR (green) and β-adaptin (blue). Arrows mark thepunctual structures containing PS1, DOR and β-adaptin (insets). Scalebar, 8 μm. (c) HEK293 cells transfected with HA-β₂AR, HA-DOR or HA-B2Rwere subjected to immuno-precipitation with buffer containing 0.1%Triton X-100 (left) or 1% CHAPSO (right), and the immunoprecipitates and5% whole cell lysates were immunoblotted with the indicated antibodies.Data are representative of at least three independent experiments. (d)HEK293 cells transfected with β₂AR or B2R were treated with 10 μM Iso or1 μM BK. The membrane fractions were subjected to fluorogenic substrateassay (*P<0.001). Con, control; Dyn K44A, dynamin II K44A; IB,immuno-blot. BK, bradykinin.

FIGS. 14 a-14 e show enhanced γ-secretase activity and Aβ production andaccelerated amyloid plaque formation in vivo, while FIGS. 14 f and 14 gshow that a selective β2-adrenergic receptor antagonist, ICI 118,551,was effective in suppressing the formation of β-amyloid plaques. (a andb) Rats were acutely injected with 2 μg norepinephrine (i.c.v.) or 0.5mg/kg clenbuterol (i.p.). Hippocampi were subjected to fluorogenicsubstrate assay (a) or rat Aβ ELISA (b), respectively (*P<0.01). (c andd) Representative amyloid plaque burdens in cortex of female (left) andmale (right) APPswe/PS1ΔE9 mice after chronic administration of drugsfor 30 days. (c) The mice were i.c.v. administered daily with saline or3 nM Iso. (d) The mice were orally administered daily with saline or 2mg/kg Cle. Scale bar, 320 μm. (e) Quantitative analysis of areas ofamyloid plaque burdens in mice from (c) and (d) (ANOVA, p<0.05). NE,norepinephrine; Sal, saline; Cle, clenbuterol. Enhanced γ-secretaseactivity and Aβ production, and accelerated amyloid plaque formation invivo. (a, b) Acute treatment of rats with norepinephrine (NE) orclenbuterol (Cle) enhanced γ-secretase activity (a) and increasedsecreted Aβ 40 and Aβ 42 levels (b) in hippocampus. *P<0.01. (cg)Cerebral amyloid plaque formation of APPswe/PS1□E9 mice chronicallyadministered of isoproterenol (c), clenbuterol (d) or ICI 118,551 (ICI)(f). Images in c, d and f: representative plaques in female (left) andmale (right) mice. In e: quantitative analysis of amyloid plaque areasin mice from c and d (*P<0.05). In g: same for mice in f (*P<0.05).

FIGS. 15 a-15 d show results from animal model studies. (a) Latencies toescape from visual platform training test. No genotype or drug effectwas found on this training (F=2.145, P=0.096). (b) Latencies to escapein hidden platform training. The control mice showed significantimpairment compared to non-transgenic mice (F=28.754, P<0.001), whereaspropranolol treatment partially ameliorate the impairment comparing tocontrol mice (F=4.571, P=0.034). Nadolol treatment showed no effect onthe impairment (F=1.192, P=0.277). (c) Percent time spent in theplatform quadrant in the probe trial. There was a significant genotypeeffect between control and non-transgenic control mice (P=0.002).Propranolol treatment partially ameliorated the impairment of spatialmemory (P=0.048). Nadolol had no effect (P=0.969).

FIGS. 16 a-16 d show results from animal test similar to those in FIG.15, but with receptor subtype specific antagonists. Betaxolol is aβ1AR-selective antagonist that can cross the BBB. ICI 118,551 is aβ2AR-selective antagonist that can cross the BBB. (a) In the visibleplatform training, no drug effect was observed (F=0.0310, P=0.969). (b)In the hidden platform training, ICI 118,551 treatment significantlyameliorated cognitive impairment (F=24.164, P<0.001). Betaxolol showedsome extent of amelioration, but the effect was not statisticallysignificant (F=3.698, P=0.057). (c) In the probe trial, the effect ofICI 118,551 was significant (P=0.005). Betaxolol showed no effect(P=0.552).

FIGS. 17 a-17 d show results from animal test similar to those in FIG.16. Metoprolol is a β1AR-selective antagonist that can cross the BBB.Butoxamine is a β2AR-selective antagonist that can cross the BBB. (a) Inthe visible platform training, no drug effect was observed (F=2.017,P=0.139). (b) In the hidden platform training, Butoxamine treatmentsignificantly ameliorated cognitive impairment (F=15.581, P<0.001).Metoprolol showed no effect (F=0.104, P=0.748). (c) In the probe trial,the effect of butoxamine was significant (P=0.020). Metoprolol showed noeffect (P=0.768).

FIG. 18 shows results from non-transgenic mice. β-adrenergic receptorantagonists showed no effect on non-transgenic mice, suggesting thattransgenes are essential. (a) no drug effect was observed in the visibleplatform training (F=2.327, P=0.077). (b) no drug effect was observed inthe hidden platform training (F=0.264, P=0.851). (c) no drug effect wasobserved in the probe trial (P=0.817).

FIG. 19 shows results from tests similar to those in FIG. 15, but withDOR antagonist Naltrindole. Naltrindole is a DOR-selective antagonistthat can cross the BBB. (a) In the visible platform training, no drugeffect was observed (F=0.754, P=0.391). (b) In the hidden platformtraining, Naltrindole treatment ameliorated cognitive impairment(F=4.945, P<0.030). (c) In the probe trial, the effect of naltrindolewas significant (P=0.006).

FIG. 20 shows escape latencies of saline (Sal), clenbuterol (Cle) andICI 118,551 (ICI) treated double-transgenic mice together with thenon-transgenic littermates (NTg) in the visible platform version ofMorris water maze (2 blocks of 4 trials each day). No significant effectof drug treatments or transgene was found for either group of mice.(n=3-7).

FIG. 21 shows escape latencies of saline (Sal), clenbuterol (Cle) andICI 118,551 (ICI) treated double-transgenic mice together with thenon-transgenic littermates (NTg) swimming to the hidden platform in theMorris water maze (1 blocks of 4 trials each day). A significanttransgene effect was seen between NTg and Sal mice. The ICI micedisplayed a faster learning curve than the Sal ones, whereas Cletreatment seemed to have no effect. (n=3-5).

FIG. 22 shows percentage time spent on the platform during the probetrial run at 24-h after the final hidden platform training in the Morriswater maze. The NTg and ICI mice spent greater proportion of time in thetarget quadrant. (n=3-5).

FIG. 23A-23C show results of surface-associated DOR receptor binding toPS1. FIG. 23A: Representative example of mixed emission spectra ofGFP-DOR donor and PS1-Cy3 acceptor fluorophores (excitation, 488-nmlaser) are taken before (red line) and after (blue line) photo-bleaching(with 561-nm laser) in HEK293 cells co-transfected with GFP-DOR andHA-ps1. Spectra are shown for one photobleached region (left plot) andanother region without photobleaching (right plot) in the same cell. GFPdonor emission increases only in the photobleached region of the cell.FIG. 23B: A set of unmixed GFP-DOR and PS1-Cy3 images of 293 cells aretaken before and after acceptor photobleaching. The region ofphotobleaching is indicated by the white outlined box. The enlargedpseudocolored images at the bottom show the intensity of GFP emission inthe photobleached and non-photobleached regions of cell surface takenbefore and after bleaching. The surface-associated intensity of donorGFP-DOR emission in 293 cells increases after acceptor ps1-Cy3photobleaching. Scale bar indicates 10 μm. FIG. 23C: Averaged FRETefficiencies (%) between surface-associated GFP-DOR and PS1-Cy3. Thenumbers in the upper the columns indicate the cells taken forexperiments. Data are from three independent experiments. **, p<0.01 ascompared with the negative control cells transfected with GFP-DOR.

DETAILED DESCRIPTION

The present invention relates to methods for screening reagents fortreating or preventing Alzheimer's disease or other related neurologicalpathologies.

Embodiments of the invention relates to treatments of Alzheimer'sdisease using antagonists of adrenergic or opioid receptors,particularly β-adrenergic or δ-opioid receptor antagonists. Someembodiments of the invention relate to new uses of β-adrenergic receptorblockers (antagonists) in the treatment of AD, and some embodiments ofthe invention relate to novel use of δ-opioid receptor (DOR) antagonistsfor treating Alzheimer's disease. Some embodiments of the inventionrelate to methods for screening reagents that can be used to treat AD orrelated neurological pathologies.

As noted above, FAD accounts for about 10% of all AD. Therefore, factorsother than genetics may play important roles in AD etiology.Environmental factors, such as stress, may exert their effects byactivating receptors, including β-adrenergic receptors (β-ARs) andδ-opioid receptor (DOR), which are G protein coupled receptors (GPCR).Several GPCRs are expressed in the central nervous systems (CNS),especially β₂-adrenergic receptor (β₂AR), are expressed in hippocampusand cortex, the main regions in the brain involved in AD pathogenesis.In the CNS, these receptors function to mediate signal transduction forepinephrine, dopamines, and opioid peptides, leading to modulation ofvarious neural functions, such as stimulus responses, learning, memory,and pain sensation.

Once activated, these receptors couple to heterotrimeric guaninenucleotide-binding proteins (G proteins) and induce downstream signalingby modulating the levels of intracellular second messengers, such ascAMP. In addition, the activated receptors also undergoclathrin-mediated endocytosis, which plays a crucial role not only inreceptor desensitization, but also in signal transduction. Theendocytosed GPCR cycles through early endosomes, late endosomes andlysosomes (LEL). The transportation of various endocytic vesicles(endosomes) is mediated by Rab GTPase, which may also serve as a markerfor various endosomes.

Embodiments of the invention are based on unexpected findings by theinventors that activation of β-adrenergic receptors (particularly,β-adrenergic receptors) or δ-opioid receptors can lead to enhancedγ-secretase in the late endosomes and lysosomes (LEL). Being an asparticprotease having an acidic optimal pH, the activity of γ-secretaseaccumulated in the LEL, which has an acidic environment, is enhanced,leading to increased production of Aβ.

FIG. 3 illustrates the path from β-adrenergic receptor or δ-opioidreceptor activation to increased production of Aβ. As shown in FIG. 3,activation of β-adrenergic receptors and δ-opioid receptors isaccompanied by clathrin-mediated endocytosis, which involves theformation of clathrin-coated pits (shown as CCP in FIG. 3) and pinchingoff of the CCP. Inventors of the present invention have found that theactive-site component of γ-secretase, presenilin-1 (shown as PS1 in FIG.3), is constitutively associated with these receptors. As a result ofsuch endocytosis, presenilin-1 or γ-secretase is brought into endosomes.Then, through vesicle trafficking mediated by Rab5 and Rab7, theseendosomes are transferred to the late endosomes and lysosomes (LEL),where the activities of γ-secretase are enhanced. The enhanced activityof γ-secretase then leads to increased production of Aβ.

These findings suggest that inhibition of β-adrenergic receptors andδ-opioid receptors by antagonists can prevent the enhanced γ-secretaseactivity. Accordingly, antagonists of these receptors may be used toreduce the production of Aβ, and hence they may be used to prevent ortreat AD or related neurological pathologies. As used herein,“antagonist” is used in a broad sense to include compounds that canprevent, reduce, or abolish receptor activation. Such compounds maycompete with the receptor agonists for the same binding site, or theymay bind to a different site to reduce the effects of agonists.

In addition, these findings suggest that potential antagonists for usein treating or preventing AD or related neurological pathologies may bescreened for by monitoring endocytosis of relevant receptors. Theendocytosis of these receptors may be determined by monitoring theendocytosis of presenilin-1 (PS1) or γ-secretase, accumulation ofpresenilin-1 or γ-secretase in LEL, enhanced activity of γ-secretase, orenhanced amyloid-β (Aβ) production.

Accordingly, some embodiments of the invention relate to methods forscreening reagents that can be used to treat or prevent AD or relatedneurological pathologies. The screening methods may be based on theabilities of candidate reagents to inhibit endocytosis of receptors thatare associated with presenilin-1 or γ-secretase or based on theirabilities to reduce or disrupt the association between the receptors andpresenilin-1 or γ-secretase. As shown in FIG. 4, a method 40 inaccordance with embodiments of the invention includes measuring theextents of endocytosis of a receptor that is associated withpresenilin-1 (or γ-secretase) or measuring the extent of associationbetween the receptor and presenilin-1 or γ-secretase, in the presenceand absence of a candidate reagent (step 41). Such receptors include Gprotein-coupled receptors that may be endogenous or from transfectionwith vectors containing genes encoding the receptors.

Then, the difference in the extents of endocytosis or association in thepresence and absence of the candidate reagent is determined (step 42).As noted above, the extent of endocytosis may be monitored byquantifying the endocytosed vesicles, endocytosed presenilin-1 orγ-secretase, accumulation of presenilin-1 or γ-secretase in LEL,enhanced γ-secretase activity in LEL, or enhanced production of Aβ. Theextent of association between the receptor and presenilin-1 org-secretase may be measured using any suitable methods, such asfluorescence energy transfer (FRET) that will be described in detaillater.

If the difference in endocytosis or association (as determined in step42) is significant or exceeds a threshold value, then the candidatereagent can potentially be used to treat or prevent AD or relatedneurological pathologies (shown as 43). If the difference isinsignificant, then the previous step may be repeated with anothercandidate reagent (shown as 44). Note that while the method in FIG. 4 isillustrated in a sequential manner, in which one candidate reagent istested at a time, one skilled in the art would appreciate that manyreagents may also be simultaneously tested, for example by using amulti-well plate or other large scale or high throughput screeningsetup.

Some embodiments of the invention relate to methods for treating orpreventing Alzheimer's disease or related neurological pathologies byadministering to a subject an effective amount of an antagonist thatbinds β-adrenergic receptor (in particular, (β2-adrenergic receptor)and/or δ-opioid receptor. The effective amount of the antagonist issufficient to reduce receptor endocytosis that starts the process ofbringing γ-secretase to the late endosomes and lysosomes (LEL). Otherembodiments of the invention relate to the use of an antagonist thatbinds β-adrenergic receptor (in particular, β2-adrenergic receptor)and/or δ-opioid receptor in the manufacturing of a medicament fortreating or preventing Alzheimer's disease or related neurologicalpathologies.

An effective amount of an antagonist that binds β-adrenergic and/orδ-opioid receptors will depend on the mode of administration, frequencyof administration, and the type of pharmaceutical composition used todeliver the compound into a patient, as well as weight, gender, age, andphysical conditions of the patient. Typically, an effective dose mayrange from about 1 μg/Kg body weight to about 10 mg/Kg body weight perday. While individual needs vary, determination of optimal range ofeffective amounts of each compound is within the skills of one skilledin the art. Administering a compound of the invention to a patient maybe via any suitable route used for administering similar pharmaceuticalsto a patient, including oral administration, injection, and transdermalpatch, to name a few.

Compounds or compositions in accordance with embodiments of theinvention may be used to treat Alzheimer's disease or relatedneurological pathologies in a mammal (human and non-human mammals). Suchcompounds or compositions of the invention may include pharmaceuticallyacceptable carriers and/or excipients, such as saline, buffer, glucose,glycerin, alcohol, starch, etc. In addition, these compounds orcompositions may be prepared in dosage forms that are commonly used forsimilar pharmaceuticals, including injections, pills, capsules, patches,etc. Methods for making these dosage forms are well known in the art.

Although various approaches to reducing the production of Aβ have beenreported, including modulation of APP production (e.g., U.S. Pat. Nos.6,187,756 and 6,043,224) and inhibiting APP processing (e.g., U.S. Pat.No. 5,242,932), embodiments of the invention are based on a differentmechanism—inhibition of receptor endocytosis that brings γ-secretase tothe LEL. The following experiments and examples clearly establish therationale for using β-adrenergic receptor (particularly, β2-adrenergicreceptor) and/or δ-opioid receptor antagonists or inhibitors to treat orprevent Alzheimer's disease or related neurological pathologies, inaccordance with embodiments of the invention.

Example 1: Amyloid β Production is Increased by Activating β₂AR

The effects of β₂AR activation on Aβ production are first assessed inHEK293 cells, which possess functional GPCR signaling pathways anddisplay normal Aβ secretion. The HEK293 cells used in this experimentwere transfected with β₂AR and mutant APP (APPswe), which harborsFAD-linked “Swedish” mutations at codons 670 and 671. As shown in FIG. 5a, stimulation of β₂AR with an agonist, isopranolol (Iso), increased thelevels of two secreted Aβ species (Aβ₄₀ and Aβ₄₂). On the other hand,addition of a β₂AR antagonist, propranolol (Pro), which has no effect onits own, abolished the ability of Iso to increase the secreted Aβlevels. The increased Aβ production requires γ-secretase, as evidencedby the fact that pre-treatment with a specific γ-secretase inhibitorL685,458 abolished the increased production of the secreted Aβ.

The fact that γ-secretase is involved in the β₂AR-induced production ofAβ is further corroborate by co-transfection of a γ-secretase substrate(C99) and β₂AR into HEK293 cells. C99 is a product ofβ-secretase-mediated cleavage of APP (see FIG. 1). C99 functions as adirect substrate of γ-secretase as well as an immediate precursor toAβ²⁶. FIG. 5 b shows that stimulation of β₂AR with Iso resulted in anincrease in Aβ production in the co-transfected HEK293 cells, similar tothe above-described results using cells co-transfected with APPswe andβ₂AR. This increase was again abolished by the presence of Pro, whichhad no effect per se. Thus, the increase in the secreted Aβ was likelydue to an enhanced γ-secretase activity.

In addition to β₂AR, activation of δ-opioid receptor (DOR) was alsofound to result in increases in secreted Aβ levels. As shown in FIG. 5c, DADLE (D-Ala₂-D-Leu₅-enkephalin, an agonist of δ-opioid receptor)treatment in C99-transfected HEK293 cells resulted in increasedproduction of Aβ. Treatment with a δ-opioid receptor antagonist, NALT(δ-naltrindole), abolished the effect of DADLE. While the aboveexperiments used cells having transfected receptors, the same resultswere seen with endogenous receptors. FIG. 5 d shows that in primaryhippocampal culture cells transfected with C99, stimulation ofendogenous β-ARs or DOR also led to an elevation of secreted Aβ.

The above results clearly indicates that activation of β-AR or DOR leadsto increased secretion of Aβ. The production of the secreted Aβ is fromcleavage of the transfected C99 substrate as shown in a pulse-chaseexperiment. As shown in FIG. 5 e, the turnover of C99 in the HEK293cells co-transfected with DOR and C99 was more rapid in the presence ofDADLE (a DOR agonist) treatment than in the absence of DADLE treatment.This result suggests that the C99 cleavage was facilitated by receptoractivation. Thus, activation of β-adrenergic receptors (β-AR)(particularly, β₂AR) and/or DOR enhances the production and secretion ofAβ as a result of promoted cleavage of C99 (or similar substrates) byγ-secretase.

Example 2: Activation of β₂AR Enhances γ-Secretase Activity

The enhanced production of Aβ upon activation of β-AR or DOR describedabove could result from increased γ-secretase expression or enhancedγ-secretase activity. To answer this question, the effect of β₂ARactivation on γ-secretase expression and activation was assessed. Asshown by Western blot analysis in FIG. 6 a, C60, the produced fromγ-secretase-mediated cleavage of C99, production was increased after Isotreatment of the C99-transfected HEK293 cells. However, the sametreatment failed to produce any change in the expression level of PS1,which is the active site component of γ-secretase and exists as aheterodimer of an amino- and a carboxyl-terminal fragments (PS1-NTF andPS1-CTF). This result suggests that β₂AR activation increasedγ-secretase activity, without changing the γ-secretase expression.

To directly measure the enzyme activity of γ-secretase, a fluorogenicsubstrate was used. The fluorogenic substrate is based on theγ-secretase-specific substrate sequence conjugated with a fluorescentreporter molecules. It was found that γ-secretase activity was enhanced30 min after stimulation of endogenous β₂AR in C6 glioma (FIG. 6 b).This effect was confirmed in acute hippocampal slices (FIG. 6 c).Presenilin-deficiency abolished the Iso-induced enhancement in mouseembryonic fibroblasts (FIG. 6 d), confirming the specificity of thisassay for γ-secretase activity. Taken together, these data clearly showthat activation of β₂AR stimulates γ-secretase activity, leading toincreased Aβ production.

The enhancement of γ-secretase activity upon activation of receptors isnot limited to β-AR. Similar results were also observed when endogenousDOR in SH-SY5Y neuroblastoma (FIG. 7 a) or in primary hippocampalcultures (FIG. 7 b) were stimulated. Furthermore, the results from thethese γ-secretase assays showed that γ-secretase activity peaked ataround 30 min and returned to the basal level at about 60 min afterreceptor (e.g., β₂AR) stimulation (FIG. 7 c).

Example 3: Enhanced γ-Secretase Activity is Independent of cAMPSignaling

As discussed above, once activated, GPCR (including β₂AR) may induce Gsprotein-dependent adenylyl cyclase activation, leading to elevatedintracellular cAMP level. To delineate the molecular mechanismresponsible for γ-secretase activity enhancement by β₂AR activation, anovel β₂AR mutant (β₂AR T68F, Y132G, Y219A or β₂AR TYY) incapable of Gsprotein activation was used in a further study. It was found that thesemutations of β₂AR failed to abolish the enhancement of γ-secretaseactivity (FIG. 8 a). This result rules out the involvement of Gs proteinsignaling in the β₂AR effect on γ-secretase. Furthermore, cells treatedwith reagents such as cholera toxin (CTX), forskolin (Fsk), anddybutyl-cAMP (db-cAMP), which mimic G protein activation and cAMP levelelevation, did not result in enhanced γ-secretase activity (FIG. 8 b).Thus, the enhanced γ-secretase activities that result from β-adrenergicreceptor activation do not involve cAMP signaling.

The fact that enhancement of γ-secretase activity does not involve cAMPsignaling suggest that this might also be true for DOR. It is known thatDOR activates pertussis toxin (PTX)-sensitive Gi/o protein, which thenleads to decreased cAMP levels by inhibiting adenylyl cyclase. It wasfound that pre-treatment of SH-SY5Y neuroblastoma with PTX did not alterthe enhancement of γ-secretase activity in response to DADLE stimulation(FIG. 9). This result suggests that enhancement of γ-secretase activityby DOR activation is also not regulated by cAMP. Therefore, regulationof γ-secretase by β₂AR or DOR activation does not rely on G proteinsignaling or the canonical cAMP pathway.

Example 4: Receptor Endocytosis Correlates with Enhanced γ-SecretaseActivity

If the enhanced γ-secretase activity does not involve G-proteinsignaling nor the cAMP pathway, then what is the mechanism? As discussedabove with reference to FIG. 3, GPCR (including βAR and opioidreceptors) activation is often accompanied by receptor endocytosis,which can also initiate specific signaling. Whether receptor endocytosisand the associated signaling are involved in the enhanced g-secretaseactivity can be probed by using various inhibitors of the endocytosispathway.

FIG. 8 c shows that the effect of Iso on γ-secretase activity can beabolished by treatment with endocytosis inhibitors, such as concanavalin(Con A), hypertonic solution treatment (Suc), and potassium-depletedmedium (K⁺ dpl). FIG. 8 d shows that the Iso-induced enhancement ofγ-secretase activity can be abolished by transfection with a dominantnegative version of dynamin (Dyn K44A) that inhibits clathrin- orcaveolin-mediated endocytosis. As β₂AR mainly internalizes in aclathrin-dependent manner, small interfering RNA (RNAi) against clathrinheavy chain may be used to deplete cellular clathrin expression. FIG. 8e shows that the Iso-induced enhancement of γ-secretase activity couldindeed by abolished by the RNAi. These results together show thatβ₂AR-induced enhancement of γ-secretase activity is mediated bysignaling mechanisms associated with agonist-induced andclathrin-mediated endocytosis.

To further confirm the requirement of agonist-induced endocytosis ofβ₂AR for γ-secretase activity enhancement, the experiments were repeatedwith another mutant of β₂AR (β₂AR L339,340 A, or β₂AR LL) as well asanother intrinsic adrenergic receptor β₃AR, both of which are deficientin agonist-induced endocytosis. It was found that stimulation of thesereceptors in HEK293 cells resulted in neither receptor endocytosis (FIG.8 f), nor enhanced γ-secretase activity (FIG. 8 g), though elevation ofthe cAMP level did occur (data not shown). These results clearly showthat agonist-induced and clathrin-mediated endocytosis of β₂AR isinvolved in the enhancement of γ-secretase activity.

The above experiments show that clathrin-mediated endocytosis isnecessary for the enhancement of γ-secretase by receptor activation.However, it is till not clear whether clathrin-mediated endocytosis byitself is insufficient to induce the enhancement of γ-secretaseactivity. To answer this question, HEK293 cells were treated withtransferrin, which can induce constitutive clathrin-dependentendocytosis of transferrin receptor. As shown in FIG. 10, transferringtreatment failed to enhance γ-secretase activity, even though it caninduce constitutive endocytosis. These results suggest thatclathrin-mediated endocytosis is necessary, but not sufficient, forβ₂AR-induced enhancement of γ-secretase activity.

Example 5: Enhanced γ-Secretase Activity and Aβ Production Associatewith Endocytic Pathway

As shown in FIG. 3, once inside the cell, the endocytic vesicles aretransported to their destinations via specific endocytic pathways. Theendocytic pathways involve trafficking of intracellular compartmentsthat are regulated by Rab guanosine triphosphatases (Rab GTPase). It isknown that endocytic transports from plasma membrane to early endosomesand then to LEL can be blocked by Rab5 S34N or Rab7 T22N, which aredominant negative mutants of early endosome marker Rab5 or LEL markerRab7, respectively. As shown in FIG. 11 a and FIG. 11 b, expression ofRab5 S34N or Rab7 T22N in HEK293 cells abolished β₂AR-stimulatedenhancement of γ-secretase activity (FIG. 11 a) and Aβ production (FIG.11 b). Since the Rab7 T22N transfected cells can still have endocyticvesicles transported to the early endosomes. These results suggest thatthe enhanced γ-secretase activity and Aβ production require theendocytic vesicles to be transported to the LEL, indicating that LEL iscritically involved in the effects of β₂AR on γ-secretase activity andAβ production.

To further show the involvement of LEL, LEL vesicles wereimmuno-isolated from Flag-Rab7-transfected cells with Flag antibody, andsubsequently verified by blotting the isolates with an early endosomemarker, early endosome antigen 1 (EEA1), or an LEL marker,lysosome-associated membrane protein-1 (LAMP-1). As shown in FIG. 11 c,the amount of Aβ, but not Flag-Rab7 or LAMP-1, in the LEL was markedlyincreased after 1-h β₂AR stimulation, indicating that Aβ production inthe LEL was enhanced by β₂AR activation without increasing the amount ofLEL.

Again, the involvement of LEL in the enhancement of the γ-secretaseactivity is not limited to β-AR. FIG. 12 shows that γ-secretase activityin the LEL was also enhanced after DOR stimulation. The experiment shownin FIG. 12 was performed with SH-SY5Y neuroblastoma. These cells weretreated with 1 μM DADLE for 30 minutes and then fractionated. Thefractions were subjected to alkaline phosphatase (AP) assay andfluorogenic substrate assay. The results clearly show that DADLEtreatment enhanced the γ-secretase activity in the AP positive fraction(*P<0.01), but not in the AP negative fraction.

Taken together, the results shown in FIG. 11 and FIG. 12 suggest thatthe LEL plays a critical role in the effects of β₂AR or DOR activationon Aβ production. These observations confirm that endocytic compartmentscan provide optimal environments for γ-secretase activity.

To further confirm that γ-secretase in the LEL is associated with theincreased Aβ production, immunofluorescence microscopy was used toexamine whether localization of γ-secretase in the LEL is promoted byβ₂AR stimulation. For experiments in cell lines, LEL was marked with theexpressed GFP-Rab7. FIG. 11 d shows that co-localization of PS1(γ-secretase active site subunit) with GFP-Rab7 occurred in HEK293 cells30 min after β₂AR stimulation in transfected HEK293 cells. For acutehippocampal slices, LEL was marked with LAMP-1 by using specificantibody. FIG. 11 e shows that co-localization of PS1 or nicastrin(another γ-secretase component) with LAMP-1 increased after Isotreatment. Together the above results suggest that β₂AR stimulationpromotes the localization of γ-secretase to the LEL, which in turn leadsto enhanced γ-secretase activity and Aβ production.

Example 6: Constitutive Association Between PS1/γ-Secretase and β₂AR

The finding that co-expression of Dyn K44A or Rab5 S34N effectivelyprevented the elevated localization of PS1 in LEL following β₂ARstimulation (FIG. 13 a) suggests that PS1 may be transported to LEL fromthe plasma membrane. Using β-adaptin, which specifically marksclathrin-coated pits and vesicles, it was found that co-localization ofPS1 with β-adaptin and the endocytosed receptor after 3-min stimulationof DOR in HEK293 cells (FIG. 13 b). These findings imply theco-endocytosis of PS1 and the activated receptor after agoniststimulation of β₂AR or DOR. This is not surprising because PS1constitutively interacts with membrane proteins, such as APP and Notch,and β₂AR can mediate the endocytosis of other transmembrane proteins byforming heterodimers with the latter. To show that this is the case, theassociation between PS1 and β₂AR or DOR was examined withco-immunoprecipitation assays.

As shown in FIG. 13 c, the four essential γ-secretase components,PS1,nicastrin, anterior pharynx defective-1a (APH-1a) and presenilinenhancer-2 (PEN-2), were co-precipitated with β₂AR or DOR in theCHAPSO-containing buffer (left panel in FIG. 13 c), in which γ-secretasestays as a complex. Replacement of CHAPSO with Triton X-100, which isknown to dissociate γ-secretase complex, disrupted the co-precipitationof receptors with nicastrin, APH-1a and PEN-2, but not PS1, as detectedwith PS1-NTF or PS1-CTF antibodies (middle panel of FIG. 13 c). Theseresults suggest that β₂AR and DOR associate with γ-secretase via directbinding to PS1. However, such associate does not occur with every GPCRbecause another member of GPCRs, B2 bradykinin receptor (B2R), failed toassociate with PS1/γ-secretase (FIG. 13 c, right) or induce γ-secretaseactivity (FIG. 13 d). Taken together, these results suggest that theβ₂AR-PS1 or DOR-PS1 association was specific and provided a mechanisticbase for the enhancement of γ-secretase activity by activation of thesereceptors. In addition, these results suggest that reagents that candisrupt or weaken receptor-PS1 associations are potential therapeuticagents for the treatment or prevention of Alzheimer or relatedneurological diseases.

Example 7: Screening of Reagents That Can Disrupt or Weaken theAssociation Between PS1 and Receptors

Reagents that can disrupt or weaken the association between PS1 andreceptors may be screened with any suitable method, such as FRET(fluorescence resonance energy transfer). FRET is a process in whichenergy is transferred from an excited donor fluorophore to an acceptorfluorophore via short-range (≦10 nm) dipole-13 dipole interactions.Thus, FRET can be used to detect physical interactions between twobinding proteins. In FRET, the non-radiative transfer of energyattenuates light emission from the donor. Thus, FRET may be detected,for example, by comparing the intensities of light emission from thedonor in the same sample before and after destroying the fluorescentmoiety on the acceptor by a suitable method, such as photo-bleaching. IfFRET is present, donor emission will be more intense afterphoto-bleaching of the acceptor.

In one example, changes in FRET efficiencies between GFP-DOR and PS1-Cy3before and after PS1-Cy3 photo-bleaching were detected in co-transfectedHEK293 cells. The cells were co-transfected with GFP-DOR and HA-ps1.HA-PS1 expression may be identified by adding a primary antibody againstHA and a second antibody conjugated with fluorophore, Cy3 (JacksonImmunoResearch), while GFP-DOR expression may be identified by GFPfluorescence.

FIGS. 23A-23C show results from one such experiment. The images, whichwere obtained with a Leica confocal microscope, include mixed emissionspectra of the GFP-DOR donor and PS1-Cy3 acceptor fluorophores(excitation with a 488 nm laser). First, it was examined whether theemission of light by the donor (GFP-DOR) became more intense afterphoto-bleaching of the acceptor (PS1-Cy3) when two proteins wereco-expressed in HEK293 cells. FIG. 23A shows images taken from thephoto-bleached and non-photo-bleached regions of the cell before andafter localized photo-bleaching. The selected regions werephoto-bleached with a 561 nm laser, which is within the absorptionspectrum of Cy3. The intensity corresponding to GFP-DOR emission wassubstantially higher in the photo-bleached region, as compared to thenon-photobleached region in the same cell. These results indicated thatFRET occurred between GFP and Cy3, and such energy transfer is abolishedor reduced upon photo-bleaching of Cy3.

FIG. 23B shows a representative set of individual images (GFP-DOR orPS1-Cy3) from the transfected HEK293 cells was illustrated before andafter photo-bleaching of the acceptor with 561 nm laser. The GFP-DORimages showed an increase in the donor emission (pseudocolored intensityimages) after photo-bleaching, and this increase occurred only in theregion of the cell exposed to the photo-bleaching.

As shown in FIG. 23C, the averaged relative FRET efficiencies betweenthe associated GFP-DOR and PS1-Cy3 near the surface region were found tobe around 22.8±3.9%, which serves as an indicator of their interaction.In the positive control, the cells expressing GFP-DOR were incubatedwith a primary antibody against GFP and a second antibody conjugatedwith Cy3. This positive control showed a FRET efficiency 27.9±5.3%. As anegative control, the cells expressing GFP-DOR were incubated with aprimary antibody against actin and a second antibody conjugated withCy3. This negative control showed a FRET efficiency 7.8±4.7%. These datatogether suggest that DOR receptor associates with PS1.

Example 8: Enhanced γ-Secretase Activity, Aβ Production, and AmyloidPlaque Formation in Animals

The effects of β₂AR on these AD-linked molecules were furtherinvestigated in animals. The in vivo experiments showed that both theγ-secretase activity (FIGS. 14 a) and Aβ levels (FIG. 14 b) in rathippocampus were significantly enhanced by acute injection of anendogenous ligand for adrenergic receptors (norepinephrine, NE) or aknown β₂AR-selective agonist clenbuterol (Cle). Based on these results,it can be expected that chronic exposure to agonists of these receptorsmay worsen AD-related pathology in animal models. Experiments in an ADmouse model (APPswe/PS1ΔE9 double-transgenic mice) support this idea, asthose mice displayed increased cerebral amyloid plaques after chronicadministration of Iso or Cle for 30 days (FIGS. 14 c-14 e). Thisobservation indicates that activation of β₂AR can enhance γ-secretaseactivity, Aβ production, and amyloid plaque formation. Therefore,antagonists of these receptors should be useful in reducing Aβ oramyloid plaque formation. FIGS. 14 f and 14 g show that this is indeedthe case, as ICI 118,551, a β2AR specific antagonist, significantlyreduced the amounts of amyloid plaques.

Example 9: In Vivo Animal Model Studies

To assess the in vivo effectiveness of β-adrenergic receptorantagonists, such compounds were administered to a transgenic mousemodel of Alzheimer's disease (APPswe/PS1ΔE9). At about 6 months of age,these mice would have progressive spatial memory deficits that areaccompanied by rising cerebral Aβ levels and increasing numbers ofcerebral amyloid plaques.

In the following studies, APPswe/PS1ΔE9 mice and non-transgenic (NTg)littermates were assigned to sex- and age-matched cohorts. Testcompounds were orally administered, beginning at 4 months of age andcontinuing for one or two months. The effects of various compounds onthe APPswe/PS1ΔE9 and non-transgenic mice were then assessed with theMorris water maze tests.

The Morris water maze test developed by neuroscientist Richard G. Morrisin 1984 is commonly used today to explore the role of hippocampus in theformation of spatial memories. The maze used in the experiments was acircular pool (diameter 1.2 m) filled with water at 24-25° C., which wasmade opaque by the addition of milk powder. The pool was placed amongfixed spatial cues consisting of boldly patterned curtains and shelvescontaining distinct objects. During the tests, mice were gently loweredinto the water, facing the wall of the pool. Mice first underwentvisible platform training for a selected number of sessions (e.g., 2consecutive days with eight trials per day), swimming to a raisedcircular platform (10 cm diameter) marked with a pole. Visible platformtrainings were split into two training blocks (e.g., four trials perday) for statistical analysis. During the visible platform training,both the platform location (NE, SE, SW, or NW) and start position (N, E,S, or W) were varied pseudo-randomly in each trial.

Hidden platform training was conducted over a selected number of days(e.g., 6 consecutive days, four trials per day), wherein mice wereallowed to search for a platform submerged 1.5 cm beneath the surface ofthe water. Mice failing to reach the platform within 60 sec were led tothe platform. During the hidden-platform trials, the location of theplatform remained constant, and mice entered the pool in one of the fourpseudo-randomly selected locations (N, E, S, or W). After each hiddenplatform trial, mice remained on the platform for 30 sec and wereremoved from the platform and returned to their home cage.

Twenty-four hours after the final hidden platform training, a probetrial was conducted in which the platform was removed from the pool andmice were allowed to search for the platform for 60 sec. All trials weremonitored by a camera mounted directly above the pool and were recordedand analyzed using a computerized tracking system.

Animal Model Experiment 1

In one experiment, propranolol and naldolol were administered toAPPswe/PS1ΔE9 transgenic and non-transgenic (NTg) mice to assess theeffects of β-adrenergic receptor antagonists on amyloid plaqueformation. APPswe/PS1ΔE9 mice and non-transgenic (NTg) littermates wereassigned to sex- and age-matched cohorts. Compounds were orallyadministered, with beginning at 4 months of age and continuing until 6months of age. Propranolol can readily cross the blood-brain barrier(BBB), and, therefore, it can antagonize β-adrenergic receptor of thecentral nervous system. On the other hand, nadolol, which is also aβ-adrenergic receptor antagonist, cannot cross the BBB. The mice weretested with spatial learning and memory using a Morris water maze.

In the Morris water maze task, mice were first subjected to two days ofvisible platform training, with two blocks of training each day. In eachblock of training, the latency of mice to find and climb onto theplatform was recorded. No genotype or drug effect was found on thistraining (P=0.101, FIG. 15 a).

Next, mice were subjected to six days of hidden platform training withone block of training each day. The control mice showed significantimpairment as compared to the non-transgenic mice (P<0.001, FIG. 15 b).While propranolol treatment partially ameliorates the impairment of thetreated mice as compared to control mice (P=0.039), nadolol treatmentshowed no effect (P=0.222).

Finally, mice were subjected to a probe trial 24 h after the finalhidden platform training. In this trial, mice were allowed to freelyswim without a platform for 1 min. The percentage of time spent by micein the platform quadrant was analyzed (FIG. 15 c). Again, there was asignificant genotype effect between transgenic mice and non-transgeniccontrol mice (P<0.001). Propranolol treatment partially ameliorated theimpairment of spatial memory in the transgenic mice (P=0.021), whilenadolol had no effect (P=0.703). While propanolol showed effects in theplatform tests, these effects did not result from difference in swimspeeds (FIG. 15 d).

The above study clearly shows that propranolol can antagonizeβ-adrenergic receptors in the central nervous system. Becausepropranolol is a non-selective β-adrenergic antagonist,subtype-selective β-adrenergic receptor antagonists are examined in vivoto determine which subtype is responsible for the above observedeffects. The subtype-selective β-adrenergic receptor antagonistsexamined include Betaxolol and ICI 118,511. Betaxolol is aβ1AR-selective antagonist that can cross the BBB. ICI 118,551 is aβ2AR-selective antagonist that can cross the BBB.

In the visible platform training, no drug effect was observed (FIG. 16a, P=0.747). However, in the hidden platform training (FIG. 16 b), ICI118,551 treatment significantly ameliorated cognitive impairment(P<0.001). Betaxolol showed some effect; however, this effect was not assignificant (P=0.071) as that of ICI 118,511. In the probe trial (FIG.16 c), the effect of ICI 118,551 was significant (P<0.001). Again,Betaxolol showed less significant effect (P=0.391), as compared to thatof ICI 118,511. Effects of ICI 118,511 did not result from changes inswim speeds (FIG. 16 d).

That β2AR selective antagonists are more effective is corroborated by afurther experiment comparing another pair of antagonists specific toβ1AR and β2AR, respectively. FIGS. 17 a-17 d show results from animaltest similar to those in FIG. 16. Metoprolol is a β1AR-selectiveantagonist that can cross the BBB. Butoxamine is a β2AR-selectiveantagonist that can cross the BBB. As shown in FIG. 17 a, in the visibleplatform training, no drug effect was observed (F=2.017, P=0.139).However, in the hidden platform training, Butoxamine treatmentsignificantly ameliorated cognitive impairment (F=15.581, P<0.001), asshown in FIG. 17 b. In contrast, metoprolol showed no effect (F=0.104,P=0.748). FIG. 17 c shows that in probe trials, the effect of butoxaminewas significant (P=0.020), whereas metoprolol had no effect (P=0.768).Again, the observed effects were not due to difference in swim speeds,as shown in FIG. 17 d.

Taken together, the above results indicate that β-adrenergic receptorantagonists target primarily central nervous β-adrenergic receptors(β-AR) to achieve the enhanced γ-secretase activity and reduced Aβproduction. Furthermore, most of the desired effects to amelioratespatial memory deficits can be achieved by inhibition of β2AR, whileinhibition of β1AR produce less effects. Thus, in accordance withembodiments of the invention, β-AR antagonists to be used for preventionor treatment of Alzheimer's disease are preferably those acting on β2AR.

In further experiments, it was found that these β-adrenergic receptorantagonists showed no effect on non-transgenic mice. As shown in FIG.18, no drug effect was observed in the visible platform training (FIG.18 a, P=0.054), the hidden platform training (FIG. 18 b, P=0.929), orthe probe trial (FIG. 18 c, P=0.940). These results suggest that whilethese β-adrenergic receptor antagonists are effective in amelioratingmemory deficits, they do not have detectable effects on “normal”subjects having no memory deficits.

FIGS. 19A-19C show results from tests similar to those in FIG. 15, butwith DOR antagonist Naltrindole. Naltrindole is a DOR-selectiveantagonist that can cross the BBB. FIG. 19 a shows that in the visibleplatform training, no drug effect was observed (F=0.754, P=0.391), andFIG. 19 b shows that in the hidden platform training, Naltrindoletreatment ameliorated cognitive impairment (F=4.945, P<0.030). In theprobe trial, the effect of naltrindole was significant (P=0.006), asshown in FIG. 19 c.

Animal Model Experiment 2

In further animal experiments, four-month old APPswe/PS1ΔE9double-transgenic mice were orally administered with saline (Sal), 2mg/kg clenbuterol (Cle, a β₂AR-selective agonist) or 1 mg/kg ICI 118,551(ICI, a β₂AR-selective antagonist) daily for 30 days. The mice were thentested with spatial learning and memory using a version of conventionalMorris water maze substantially as described above.

The water maze was a circular 1.2 m pool filled with water at 24-25° C.and made opaque by the addition of milk powder. The pool was placed amidfixed spatial cues consisting of boldly patterned curtains and shelvescontaining distinct objects. Mice were gently lowered into the waterfacing the wall of the pool. Mice first underwent visible platformtraining for 2 consecutive days (eight trials per day), swimming to araised circular platform (10 cm of diameter) marked with a pole. Visibleplatform days were split into two training blocks of four trials forstatistical analysis. During visible platform training, both theplatform location (NE, SE, SW, or NW) and start position (N, E, S, or W)were varied pseudo-randomly in each trial.

As shown in FIG. 20, escape latencies of saline (Sal), clenbuterol(cle), and ICI 118,551 (ICI) treated double transgenic mice togetherwith the non-transgenic littermates (NTg) in the visual platform versionof Morris water maze exhibit small, but statistically insignificant,effects of drug treatments. Similarly, a small, but statisticallyinsignificant, effect is seen between the Sal treated transgenic andnon-transgenic mice. (n=3-7).

Hidden platform training was conducted over 6 consecutive days (fourtrials per day), wherein mice were allowed to search for a platformsubmerged 1.5 cm beneath the surface of the water. Mice failing to reachthe platform within 60 sec were led to the platform. Duringhidden-platform trials, the location of the platform remained constant,and mice entered the pool in one of the four pseudo-randomly selectedlocations (N, E, S, or W). After each hidden platform trial, miceremained on the platform for 30 sec and were removed from the platformand returned to their home cage.

FIG. 21 shows escape latencies of saline (Sal), clenbuterol (Cle) andICI 118,551 (ICI) treated double-transgenic mice together with thenon-transgenic littermates (NTg), swimming to a hidden platform in aMorris water maze (1 blocks of 4 trials each day). In this test, asignificant transgenic effect was seen between NTg and Sal mice. The ICImice displayed a faster learning curve than the Sal mice, whereas Cletreatment seemed to have no effect. (n=3-5). This results suggests thatICI treatment is quite effective in ameliorating the memory deficits ofthe transgenic mice.

Twenty-four hours after the final hidden platform training, a probetrial was conducted in which the platform was removed from the pool andmice were allowed to search for the platform for 60 sec. All trials weremonitored by a camera mounted directly above the pool and were recordedand analyzed using a computerized tracking system.

FIG. 22 shows percentage time spent on the platform during a probe trialrun at 24-h after the final hidden platform training in a Morris watermaze. The NTg and ICI mice spent greater proportion of time in theplatform. (n=3-5), suggesting that ICI treatment conferred substantialeffects in ameliorating the memory deficits.

Methods and Reagents

The following describes some specific procedures used in the experimentsand examples discussed above. In the interest of clarity, generalbiochemical and molecular biology techniques used, which are well knownto one skilled in the art, will not be described.

Reagents and Cell Transfection

All reagents were from Sigma or other commercial sources, unlessotherwise indicated. Human FL-APP was cloned into pcDNA3 vector andmutated by PCR into the APPswe. The C99 with the signal peptide of APPwas subcloned into pcDNA3 vector. The authenticity of the DNA sequenceswas confirmed by sequencing. The RNAi plasmid for human clathrin heavychain was designed to target the sequence of5′-GCTGGGAAAACTCTTCAGATT-3′. The NS RNAi was5′-GGCCGCAAAGACCTTGTCCTTA-3′.

Animals and Drug Treatments

All animal experiments were conducted strictly in accordance with theNational Institutes of Heath Guidelines for the Care and Use ofLaboratory Animals. In acute experiments, Sprague-Dawly rats (fromShanghai SLAC Laboratory Animal Company) were injectedintracerebroventricularly with 2 μg norepinephrine. The stereotaxiccoordinates were: anterior-posterior, −0.9 mm; left-right, −1.5 mm;dorsal-ventral, −3.8 mm. Acute intraperitoneal injection of rats wasperformed with 0.5 mg/kg clenbuterol. In 30-d chronic experiments,APPswe/PS1ΔE9 mice (Jackson Laboratory) up to 5 months of age wereinstrumented with cannulae (anterior-posterior, 0.6 mm; left-right, 1.2mm; dorsal-ventral, 1.8 mm) for daily injections of saline (n=4, twofemales and two males) and 3 nM isoproterenol (n=6, four females and twomales). APPswe/PS1ΔE9 mice were daily subjected to oral administrationof saline (n=6, three females and three males), 2 mg/kg per dclenbuterol (n=7, four females and three males), and 1 mg/kg per d ICI118,551 (n=6, three females and three males).

Immunohistochemistry and Quantification of Amyloid Plaques

Mice were anesthetized and sacrificed by transcardiac saline perfusion.Brains were isolated, and the forebrain bisected midsagittally and wasplaced in 4% paraformaldehyde in phosphate buffer (pH 7.6) for 5 h at 4°C. Then, the post-fixed hemispheres were cryosectioned coronally into 10μm sections. The sections were incubated with anti-Aβ 6E10 antibody(Chemicon), followed by TRITC-conjugated anti-mouse antibody incubation.The sections were then visualized and imaged using a laser confocalfluorescence microscope (Leica TCS SP2). Area of amyloid plaques wasquantified with Image-Pro Plus 5.1 software (Media Cybernetic). Imageswere converted to gray scale by thresholding, and the area plaque wasestimated.

Hippocampal Cultures and Acute Slices Preparation

Primary hippocampal cultures were prepared from 1-day-postnatal SD rats,electroporated using Amaxa Nucleofector system, maintained inB27/neurobasal medium (Invitrogen) for two weeks, and then used foragonist treatment. Acute hippocampal slices from 8-week-postnatal SDrats were prepared using a vibratome in ice-cold artificial CSF.

ELISA for Aβ

Cells were exposed to Iso (10 μM) or DADLE (1 μM) for 1 h and werefurther incubated in the conditioned medium for another 6 h. Theconditioned medium was detected for Aβ₄₀ and Aβ₄₂ with sandwich ELISAkits (Biosource). Rat hippocampi were homogenized and centrifuged at100,000×g for 1 h. Supernatants were detected for rat Aβ₄₀ and Aβ₄₂ withthe BNT77/BA27 and BNT77/BC05 sandwich ELISA kits (Wako) according toprevious reports⁵². All measurements were performed in duplicate.

Immunoprecipitation

HEK293 cells or rat hippocampus slices were lysed in RIPA buffer.Flag-tagged receptors and endogenous DOR were immunoprecipitated withFlag antibody-conjugated beads or DOR antibody (Santa Cruz). Theimmunoprecipitated complexes were separated by SDS-PAGE and blotted withFlag, PS1-NTF, PS1-CTF, nicastrin, APH-1a and PEN-2 antibodies(Calbiochem). HEK293

Expressed Substrate Assay

The experiments were performed similarly as described previously²⁷.HEK293 cells were lysed and aliquots (containing 50 μg proteins) werecentrifuged at 13,000×g for 15 min. The cell membrane fractions wereresuspended and then incubated at 37° C. for 2 h in 50 μl of assaybuffer (pH 6.5) containing 1, 10-phenanthroline, aprotinin, andleupeptin. The C60 generated from the incubated membrane fractions wasmeasured by Western blot using HA antibody 12CA5.

Fluorogenic Substrate Assay

The assay was performed as reported^(28, 29). Cells or hippocampaltissues were lysed or homogenized. Aliquots (containing 50 μg celllysates or hippocampal homogenates) were centrifuged at 13,000×g for 15min. The membrane pellets were resuspended and incubated at 37° C. for 2h in 50 μl of assay buffer (pH 6.5) containing 12 μM fluorogenicsubstrates (Calbiochem). After incubation, fluorescence was measuredusing a spectrometer with excitation wavelength at 355 nm and emissionwavelength at 440 nm.

Pulse-Chase Assay

HEK293 cells cotransfected with HA-C99 and DOR were starved for 2 h inmethionine and serum free medium (Invitrogen) and subsequentlypulse-labeled with 500 μCi [³⁵S]methionine (Amersham Pharmacia) in theabsence or presence of DADLE for 1 h. The cells were then chased for 3 hin medium containing excess amounts of unlabeled methionine. The C99 incell lysates was immunoprecipitated with 12CA5 and analyzed byautoradiography.

Immuno-Isolation of LEL

The experiments were modified from vesicle isolation protocol⁵³. Thehomogenates from HEK293 cells cotransfected with β₂AR, C99 and Flag-Rab7were centrifuged at 500×g for 10 min. The resulting supernatants wereincubated at 4° C. with M2 antibody-conjugated beads for 8 h. Theisolated LEL were then subjected to Western blot with antibodies againstLAMP-1 and EEA1 (BD Biosciences).

Immunofluorescence Microscopy

For experiments in HEK293 cells transfected with HA-tagged receptortogether with or without GFP-Rab7 or GFP-Rab7 T22N, cells were fed withantibody 12CA5 for 30 min, treated with agonists, and then fixed. Forthe experiments in cells transfected with Flag-Rab7 and HA-Dyn K44A orGFP-Rab5 S34N, cells were treated with agonists and then fixed. Forexperiments in acute hippocampal slices, slices that exposed to Iso orDADLE were fixed and cryosectioned. The subsequent immuno-staininginvolved primary antibodies (including PS1-NTF, Flag, LAMP-1, orβ-adaptin antibodies or FITC-conjugated HA antibody) and secondaryantibodies (Cy3-conjugated anti-rabbit and FITC-conjugated anti-mouseantibodies, Jackson ImmunoResearch). Images were acquired using a laserconfocal fluorescence microscope (Leica TCS SP2).

FRET Measurements of Receptor-PS1 Association

Images, with or without photo-bleaching, for FRET analysis were acquiredwith a Leica TCS SP2 confocal microscope and analyzed with software.Briefly, HEK293 cells were co-transfected with GFP-DOR and HA-ps1.HA-PS1 expression was detected by monitoring Cy3 fluorescence afterincubation with a primary antibody against HA and a second antibodyconjugated with Cy3 (Jackson ImmunoResearch), while GFP-DOR expressionwas identified by GFP fluorescence detection. Emission spectra from thecells expressing GFP-DOR or PS1-Cy3 were obtained with the λ mode, usinga 488 nm laser. For measurements of FRET efficiency by this method,Leica software application for acceptor photobleaching was applied. Theselected cell surface areas were photobleached with a 561 nm laser,which bleached the Cy3 fluorophore. Reduction of the Cy3 signal afterphoto-bleaching in the GFP-DOR and HA-ps1 co-transfected HEK293 cellswas 84±5.3% (n=50) on average. FRET was resolved as an increase in theGFP-DOR (donor) signal after photo-bleaching of ps1-Cy3 (acceptor).Relative FRET efficiency was calculated as (1-[Cy3 Ipre-bleach/Cy3Ipost-bleach])×100%. For control purposes, an area of the cell surfacewithout photo-bleaching was also analyzed for FRET.

Statistical Analysis

Data from cell experiments were analyzed by Student's t-test forcomparison of independent means, with pooled estimates of commonvariances. The statistical significance of mouse data was determined byANOVA followed by student's t-test.

While the invention has been described with respect to a limited numberof embodiments, those skilled in the art, having benefit of thisdisclosure, will appreciate that other embodiments can be devised whichdo not depart from the scope of the invention as disclosed herein.Accordingly, the scope of the invention should be limited only by theattached claims.

1. A method for treating Alzheimer's disease or related neurologicalpathology, comprising administering a receptor antagonist to a subjectin need thereof, wherein the receptor antagonist inhibits endocytosis ofa G-protein coupled receptor that associates with presenilin-1 duringendocytosis.
 2. A method for treating Alzheimer's disease or relatedneurological pathology, comprising administering a reagent to a subjectin need thereof, wherein the reagent interferes with association of aG-protein coupled receptor and presenilin-1 or γ-secretase.
 3. Themethod of claim 1, wherein the G-protein coupled receptor is at leastone selected from the group consisting of a β-adrenergic receptor and aδ-opioid receptor.
 4. The method of claim 3, herein the β-adrenergicreceptor is a type 2 β-adrenergic receptor (β2AR).
 5. The method ofclaim 1, wherein the antagonist is at least one selected from ICI118,551, propranolol, butoxamine, and naltrindole.
 6. The method ofclaim 1, wherein the antagonist is ICI 118,551 or butoxamine.
 7. Amethod for preparing a reagent for treating Alzheimer's disease orrelated neurological pathology, comprising the following steps: (a)activating a receptor and determining a first extent of endocytosis ofthe receptor, wherein the receptor is a G-protein coupled receptor thatassociates with presenilin-1; (b) activating the receptor under the sameconditions as in step (a), in the presence of a candidate reagent, anddetermining a second extent of endocytosis of the receptor; (c)determining a difference between the first extent of endocytosis and thesecond extent of endocytosis; (d) repeating steps (a)-(c), if thedifference is less than a threshold; and (e) preparing and/or purifyingthe candidate reagent as a medicament for treating or preventingAlzheimer's disease or related neurological pathology.
 8. The method ofclaim 7, wherein the receptor is at least one selected from the groupconsisting of a β-adrenergic receptor and δ-opioid receptor.
 9. Themethod in accordance with claim 8, wherein the β-adrenergic receptor isa type 2 β-adrenergic receptor (β2AR).
 10. The method of claim 7,wherein the receptor is expressed on a cell that has been transfectedwith a gene encoding the receptor.
 11. The method of claim 7, whereinthe determining the first extend of endocytosis and the determining thesecond extent of endocytosis are performed by monitoring amounts ofendocytosed vesicles, presenilin-1 in endocytosed vesicles, γ-secretaseactivity in late endosomes or lysosomes (LEL), or Amyloid-beta (Aβ)formation.
 12. A method for screening a reagent for treating Alzheimer'sdisease or related neurological pathology, comprising the followingsteps: (a) activating a receptor and determining a first extent ofendocytosis of the receptor, wherein the receptor is a G-protein coupledreceptor that associates with presenilin-1; (b) activating the receptorunder the same conditions as in step (a), in the presence of a candidatereagent, and determining a second extent of endocytosis of the receptor;(c) determining a difference between the first extent of endocytosis andthe second extent of endocytosis; and (d) repeating steps (a)-(c), ifthe difference is less than a threshold.
 13. The method of claim 12,wherein the receptor is at least one selected from the group consistingof a β-adrenergic receptor and δ-opioid receptor.
 14. The method inaccordance with claim 13, wherein the β-adrenergic receptor is a type 2β-adrenergic receptor (β2AR).
 15. The method of claim 12, wherein thereceptor is expressed on a cell that has been transfected with a geneencoding the receptor.
 16. The method of claim 12, wherein thedetermining the first extend of endocytosis and the determining thesecond extent of endocytosis are performed by monitoring amounts ofendocytosed vesicles, presenilin-1 in endocytosed vesicles, γ-secretaseactivity in late endosomes or lysosomes (LEL), or Amyloid-beta (Aβ)formation.